We plan to study the mechanisms underlying the generation of antibody diversity by studies of the nucleotide sequence organization in the mRNAs and genes coding for the heavy chain variable (V sub H) regions of immunoglobulins exhibiting identical or closely related antigenic specificity. Similarities and differences in the coding and non-coding regions of the genes, and particularly those sequences surrounding the genes, will provide clues as to the origin of antibody diversity, i.e., how various V-regions are moved next to the origin of C-regions. Messenger RNAs isolated from hybridomas producing anti-influenza hemagglutinin A antibodies of the isotype gamma 2b, which show similar or identical reactivity patterns with respect to viral variants, will be employed to generate cDNAs and the latter will be cloned. Full-length cDNA clones will be detected with a gamma 2b constant region probe that we have prepared from a non-full-length previously cloned cDNA. Restriction fragments obtained from the 5' end of a cloned full-length cDNA provides V-region specific probes. Bacteriophage containing gene libraries will be constructed from each hybridoma line. The libraries will be screened and the DNA inserts of positive recombinant clones characterized with the V and C region specific probes. The nucleotide sequence organization of the cDNAs and genes will be determined and compared among the selected hybridomas which secrete antibody molecules with related combining sites. Our efforts will be concentrated in this unique system upon the specific heavy chain variable region (V sub H) nucleotide sequences because of the paucity of information regarding specific V sub H region nucleotide sequences, the importance of V sub H regions in determining antibody specificity, and the potential utility of V sub H region probes for the mRNAs and genes encoding T-cell receptors in the future.